Cadherins as Cancer Biomarkers

ABSTRACT

Methods of isolating, enriching, capturing, identifying, or detecting the presence of, cancerous cells in a sample, e.g., a blood sample from a subject, by detecting the presence of one or more cancer cell surface markers selected from the group consisting of cadherin 1 (CDH1), CDH2, CDH3, CDH4, CDH5, CDH9, CDH11, CDH17, CDH19, protocadherin 9 (PCDH9) and/or PCDH beta 13 (PCDHb13), and optionally an additional cancer cell surface marker, e.g., EpCAM, MUC1, EphB4, EGFR, CEA, and/or HER2.

CLAIM OF PRIORITY

This application is a divisional of U.S. patent application Ser. No.14/347,898, filed Mar. 27, 2014, which is a U.S. National PhaseApplication under 35 U.S.C. §371 of International Patent Application No.PCT/US2012/058069, filed on Sep. 28, 2012, which claims the benefit ofU.S. Provisional Patent Application Ser. No. 61/540,380, filed on Sep.28, 2011; the entire contents of the foregoing are hereby incorporatedby reference.

TECHNICAL FIELD

This invention relates to methods of isolating, identifying, ordetecting the presence of, cancerous cells in a sample, by detecting thepresence of one or more cancer cell surface markers selected from thegroup consisting of cadherin 1 (CDH1), CDH2, CDH3, CDH4, CDH5, CDH9,CDH11, CDH17, CDH19, protocadherin 9 (PCDH9) and/or PCDH beta 13(PCDHb13), optionally with the addition of one or more other cancer cellsurface markers, e.g., EpCAM, MUC1, EphB4, EGFR, CEA, and/or HER2.

BACKGROUND

Circulating tumor cells (CTCs) offer a non-invasive means to seriallysample tumor cells from patients with solid malignancies. Most currentCTC capture devices rely on immunoaffinity using antibodies directedagainst epithelial cell adhesion molecule (EpCAM). This particularcapture epitope is attractive for its presence in a wide variety ofepithelial cancers, but relative absence in normal blood cells. Thisprovides a strategy to specifically capture tumor cells in the blood ofpatients. However, there are variations of EpCAM levels between tumors,so a multiple epitope strategy could be more inclusive. Furthermore,there is indication that EpCAM is lost during the process of epithelialto mesenchymal transition (EMT), which has been implicated inmetastasis. Therefore, the identification of novel CTC capture epitopesto deal with tumor epitope heterogeneity as well as potentialbiologically important changes in tumor cell surface epitopes isimportant to capture the full spectrum of CTCs.

SUMMARY

The present invention is based, at least in part, on the discovery thatcertain cadherins are selectively expressed on CTCs and thus can be usedto detect, identify, isolate, and monitor the presence of CTCs in asample.

Thus in one aspect, the invention features methods for detecting thepresence of a circulating tumor cell (CTC) in a sample from a subject.The methods include providing a sample comprising blood from a subject;and contacting the sample with one or more agents that bind to a cancercell surface marker selected from the group consisting of cadherin 1(CDH1), CDH2, CDH3, CDH4, CDH5, CDH9, CDH11, CDH17, CDH19, protocadherin9 (PCDH9) and/or PCDH beta 13 (PCDHb13); and detecting the binding ofthe agent to a cancer cell surface marker present on a cell in thesample; thereby detecting the presence of a cancer cell in the sample.

In a further aspect, the invention features method of capturing (e.g.,isolating and/or enriching) a circulating tumor cell (CTC) from asample. The methods include providing a sample comprising blood from asubject; and contacting the sample with one or more agents that bind toa cancer cell surface marker selected from the group consisting ofcadherin 1 (CDH1), CDH2, CDH3, CDH4, CDH5, CDH9, CDH11, CDH17, CDH19,protocadherin 9 (PCDH9) and/or PCDH beta 13 (PCDHb13), under conditionssufficient to allow binding of the agent to cells expressing the surfacemarker; and isolating a cell expressing a cancer cell surface markerfrom the sample; thereby capturing a CTC from the sample.

In an additional aspect, the invention features methods for diagnosingcancer in a subject. The methods include providing a sample comprisingblood from the subject; contacting the sample with one or more agentsthat bind to a cancer cell surface marker selected from the groupconsisting of cadherin 1 (CDH1), CDH2, CDH3, CDH4, CDH5, CDH9, CDH11,CDH17, CDH19, protocadherin 9 (PCDH9) and/or PCDH beta 13 (PCDHb13),under conditions sufficient to allow binding of the agent to cellsexpressing the surface marker; and detecting the binding of the agent toa cancer cell surface marker present on a cell in the sample; whereinthe presence of binding to a cell in the sample indicates that thesubject has cancer.

In yet another aspect, the invention features methods for monitoringdevelopment or progression of cancer in a subject. The methods includeproviding a first sample comprising blood from the subject; contactingthe first sample with one or more agents that bind to a cancer cellsurface marker selected from the group consisting of cadherin 1 (CDH1),CDH2, CDH3, CDH4, CDH5, CDH9, CDH11, CDH17, CDH19, protocadherin 9(PCDH9) and/or PCDH beta 13 (PCDHb13), under conditions sufficient toallow binding of the agent to cells expressing the surface marker;detecting the presence or absence of binding of the agent to a cancercell surface marker present on a cell in the first sample; providing asubsequent sample comprising blood from the subject; contacting thesubsequent sample with one or more agents that bind to a cancer cellsurface marker selected from the group consisting of cadherin 1 (CDH1),CDH2, CDH3, CDH4, CDH5, CDH9, CDH11, CDH17, CDH19, protocadherin 9(PCDH9) and/or PCDH beta 13 (PCDHb13), under conditions sufficient toallow binding of the agent to cells expressing the surface marker; anddetecting the presence or absence of binding of the agent to a cancercell surface marker present on a cell in the subsequent sample.

In some embodiments, the surface markers include cadherins 1, 2, 3, 5,and 11. In some embodiments, the surface markers include cadherins 1, 2,3, 5, 11, and EpCAM. In some embodiments, the surface markers includecadherins 3 and 11. In some embodiments, the surface markers includecadherins 3, 11, and EpCAM.

In some embodiments of the methods described herein, the absence ofbinding to a cell in both the first and subsequence sample indicatesthat the subject has not developed cancer. In some embodiments of themethods described herein, the absence of binding to a cell in the firstsample, and the presence of binding to a cell in a subsequence sampleindicates that the subject has developed cancer.

In some embodiments, the methods described herein include quantifying alevel of surface marker-expressing cells in the first and subsequentsamples, wherein an increase in the number of cells that express the oneor more surface markers indicates that cancer is progressing in thesubject; a decrease in the number of cells that express the one or moresurface markers indicates that cancer is regressing in the subject; andno significant change in the number of cells that express the one ormore surface markers indicates that cancer is stable in the subject.

In some embodiments of the methods described herein, the one or moreagents that bind to a cancer cell surface marker are in a microfluidicdevice.

In some embodiments of the methods described herein, the one or moreagents that bind to a cancer cell surface marker are coated on asurface, e.g., a bead, post or obstacle, e.g., a magnetic bead.

In some embodiments of the methods described herein, the one or moreagents that bind to a cancer cell surface marker are antibodies orantigen-binding fragments thereof.

In some embodiments of the methods described herein, wherein the one ormore agents that bind to a cancer cell surface marker bind to CDH4 orCDH9.

In some embodiments of the methods described herein, the method isperformed using a microfluidic device. In some embodiments of themethods described herein, the microfluidic device separates tumor cellsbased on inertial lift forces or fluid flow patterns.

In a further aspect the invention provides devices for isolation,enrichment, separation, or detection of cells, e.g., tumor cells, in asample. The devices comprise an inlet, and outlet, and one or more areascoated with an agent that binds a cancer cell surface marker selectedfrom the group consisting of cadherin 1 (CDH1), CDH2, CDH3, CDH4, CDH5,CDH9, CDH11, CDH17, CDH19, protocadherin 9 (PCDH9) and/or PCDH beta 13(PCDHb13), configured to separate, isolate, enrich, or detect cells thatexpress the cancer cell surface marker, e.g., from cells that do notexpress the cancer cell surface marker. In some embodiments, the surfacemarkers include cadherins 1, 2, 3, 5, and 11. In some embodiments, thesurface markers include cadherins 1, 2, 3, 5, 11, and 17. In someembodiments, the surface markers include cadherins 3 and 11. In someembodiments, the surface markers include cadherins 3, 11, and EpCAM.

In some embodiments of the methods described herein, the device is amicrofluidic device. In some embodiments of the methods describedherein, the device comprises a micro-channel disposed between the inletand the outlet, and a herringbone pattern formed by arranging grooves inthe micro-channel, and the agent that binds the cancer cell surfacemarker is disposed on one or more of walls of the microchannel.

In some embodiments, the cancer is of epithelial origin, or ofmesenchymal origin.

In general, the methods described herein are in vitro methods.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a set of three images showing the results of Western blotanalysis of EpCAM, CDH2 and CDH3 expression using a commerciallyavailable antibodies (Veridex, eBio, and BD, respectively) in ten cancercell lines (HCT116, SW620, CaLu-1, HCC827, H1650, PC3-9, LBX-1, MCF10A,MDA-MB-231, and SKBR3). CaLu1, PC3-9, and LBX1, which are negative forthe EpCAM epitope, are positive for CDH2. Band is at approximately 100kD. This combination of antibodies was able to capture all 10 cell linestested, including both mesenchymal- and epithelial-derived tumor cells.

FIG. 2 is a table showing the results of analysis of candidate cadherinsusing Oncomine™ expression database.

FIGS. 3-49 are box plots showing the levels of expression of variouscell surface markers: FIG. 3, EpCAM; FIGS. 4-5, CDH1; FIGS. 6-7, CDH2;FIG. 8, CDH3; FIGS. 9-12, CDH4; FIG. 13, CDH5; FIGS. 14-18, CDH6; FIGS.19-20, CDH7; FIGS. 21-22, CDH8; FIG. 23, CDH9; FIG. 24, CDH10; FIGS.25-27, CDH11; FIG. 28, CDH12; FIG. 29, CDH13; FIGS. 30-31, CDH15; FIG.32, CDH16; FIG. 33, CDH17; FIG. 34, CDH18; FIGS. 35-36, CDH19; FIG. 37,CDH20; FIGS. 38-39, CDH22; FIGS. 40-43, CDH23; FIGS. 44-45, CDH24; andFIGS. 46-49, CDH26.

FIGS. 50A-D are bar graphs showing expression in normal breastepithelial cells (NBE), CD44+ cells isolated from pleural effusions(P_CD44+) of human breast cancer patients, and CD44+ (X_CD44+) and CD44−(X_CD44−) cells isolated from tumor xenografts grown in mice of CDH1(50A), CDH3 (50B), CDH11 (50C), and CDH17 (50D).

FIGS. 51A-F are line graphs showing the effect of treatment withTGF-beta on epithelial cells, showing that expression of some cadherinsincreases, e.g., CDH2 (51B) and CDH11 (51E); some have decreasedexpression, e.g., CDH1 (51A) and CDH17 (51F); and some do not change,3.g., CDH3 (51C) and CDH5 (51D) during EMT.

FIGS. 52A-B are line graphs showing that while Abcam 89900 anti-CDH3antibodies bound robustly to lung cancer H1650 cells (FIG. 52A, leftpanel), and RnD 1790 anti-CDH11 antibodies bound to PC3-9 prostatecancer cells (FIG. 52B, left panel), neither antibody boundsignificantly to white blood cells (52A-B, middle panels) or whole blood(52A-B, right panels).

FIG. 53 is a line graph showing the ability of a combination of CDH3 andCDH11 antibodies to capture cancer cells in a mixture of cells. Calu1breast cancer cells and H1650 lung cancer cells were mixed and contactedwith anti-CDH3 Abcam 89900 antibodies and RnD anti-CDH11 1790antibodies. This combination was extremely effective in capturing nearlyall of the cancer cells.

DETAILED DESCRIPTION

As described herein, certain cadherins are substantially absent fromnormal blood cells, but are present on a variety of cancerous cell.Thus, specific cadherins would not be detectable in a sample that doesnot contain cancer cells but would be detectable in a cancercell-containing sample. This allows for detecting, identifying,isolating, and monitoring the presence of CTCs in a sample, e.g., fordiagnosis or monitoring of cancer or therapy, based on the presence ofcadherin-expressing cells in a sample (e.g., the presence of cellsexpressing one or more cadherins at a level above a threshold, e.g., areference level or detectable level).

Methods

The methods described herein are based on using cell-surface expressionof one or more cancer cell surface markers, e.g., cadherins, fordetecting, identifying, isolating, and monitoring the presence of CTCsin a sample. The methods include the detection of one or more cadherins,as described herein, and optionally one or more additional cancer cellsurface markers, e.g., EpCAM, MUC1, EphB4, EGFR, CEA, and/or HER2.

Detecting Presence of CTCs

In some aspects, the methods described herein are used simply to detectthe presence of CTCs in a sample. The sample is contacted with acadherin binding agent as described herein, and binding to cells thathave cell-surface expression of cadherin is measured. As certaincadherins are absent in normal blood, the detection of such bindingindicates the presence of tumor cells in the sample, allowing adiagnosis of cancer in the subject. In some embodiments, the methodsinclude detecting the presence of cadherin-expressing cells in a sample(e.g., the presence of cells expressing one or more cadherins at a levelabove a threshold, e.g., a reference level or detectable level).

Capturing CTCs

The methods described herein can also include isolating or purifying theCTCs, or “capturing” CTCs, from a sample, e.g., to produce a sample thatis enriched in CTCs. The methods include contacting the sample with acadherin binding agent, and capturing cells that bind to the agent. Forexample, the methods can be performed using microfluidic devices,arrays, or beads as described herein. A sample that is enriched in CTCsis one that is at least 1% pure CTCs, in terms of percent of cells inthe sample (i.e., the cells in the sample are at least 20% CTCs). Insome embodiments, the enriched sample is at least 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, or 10% or more CTCs. The enriched samples can then be usedfor further analysis, e.g., for the diagnosis of cancer in a subject asdescribed herein.

Monitoring Progression or Development of Cancer

The methods described herein can also be used for monitoring developmentprogress of cancer in a subject. For example, in a subject who has notumors, the methods can be repeated over time; the appearance of cellsexpressing surface cadherin indicates that the subject has developedcancer. In a subject who has a benign tumor (and thus no CTCs in abaseline sample), the appearance of cadherin-expressing cells indicatesthat the subject has developed cancer, e.g., the benign tumor may havebecome malignant/metastasized.

Samples

Samples useful in the methods described herein include samplescomprising bodily fluid from a subject, e.g., blood. In someembodiments, a sample comprising whole blood is used. In someembodiments, red and/or white blood cells are removed from the samplebefore performing the methods described herein, e.g., using methodsknown in the art; such methods must selectively remove red and/or whiteblood cells, leaving any tumor cells present in the sample. A sample canbe obtained from a subject using any method known in the art, e.g.,venipuncture with a needle large enough to maintain cell integrity.

Cancer Cell Surface Markers

Cadherins are surface antigens that have been implicated in a widevariety of cancers; see, e.g. Berx and van Roy, Cold Spring HarbPerspect Biol, 1(6):a003129 (2009); Kim et al., Cancer, 110(12):2785-92(2007); Peinado et al. Int J Dev Biol, 48(5-6):365-75 (2004), and offera source of new CTC capture epitopes. Additional cancer cell surfacemarkers have been identified and can be used in the methods describedherein. See, e.g., Table 1 of Man et al., J Clinic Experiment Pathol1:102 (2011); doi:10.4172/2161-0681.1000102, and references citedtherein. For example, EpCAM is present on a wide variety of epithelialcancers (carcinomas, e.g., lung, prostate, colon, breast, bladder), butrelatively absent in normal blood cells. MUC-1 is present on colorectal,ovarian, breast, and prostate cancer cells.

A number of cadherins are useful in the methods and devices describedherein. Table A provides a list of cadherins that can be used, withGenBank Accession Nos. for human nucleic acid and protein sequences;Table B provides a list of additional cancer cell surface markers thatcan optionally be used in addition to the cadherins. One of skill in theart will appreciate that, when detecting cancer in subjects of otherspecies, sequences from those species should be used; such sequences areknown in the art and can be obtained using routine bioinformatics.

TABLE A Cadherin Cancer Cell Surface Markers GenBank Accession Nos. GeneSymbol Official Full Name Nucleic Acid (mRNA) Protein CDH1 cadherin 1,type 1, NM_004360.3 NP_004351.1 E-cadherin (epithelial) CDH2 cadherin 2,type 1, NM_001792.3 NP_001783.2 N-cadherin (neuronal) CDH3 cadherin 3,type 1, NM_001793.4 NP_001784.2 P-cadherin (placental) CDH4 cadherin 4,type 1, NM_001794.2 NP_001785.2 R-cadherin (retinal) CDH5 cadherin 5,type 2 NM_001795.3 NP_001786.2 (vascular endothelium) CDH9 cadherin 9,type 2 NM_016279.3 NP_057363.3 (T1-cadherin) CDH11 cadherin 11, type 2,NM_001797.2 NP_001788.2 OB-cadherin (osteoblast) CDH17 cadherin 17, LINM_004063.3 (v1) NP_004054.3 (v1) cadherin (liver- NM_001144663.1 (v2)NP_001138135.1 (v2) intestine) CDH19 cadherin 19, type 2 NM_021153.2NP_066976.1 PCDH9 protocadherin 9 NM_203487.2 (v1) NP_982354.1 (v1)NM_020403.4 (v2) NP_065136.1 (2) PCDHB13 protocadherin beta NM_018933.2NP_061756.1 13

TABLE B Additional Cancer Cell Surface Markers GenBank Accession Nos.Gene Nucleic Acid Symbol Official Full Name (mRNA) Protein EPCAMepithelial cell NM_002354.2 NP_002345.2 adhesion molecule MUC-1 mucin 1,cell NM_002456.5* NP_002447.4* surface associated HER2 v-erb-b2 Isoforma: NP_004439.2 erythroblastic NM_004448.2 NP_001005862.1 leukemia viralIsoform b: oncogene homolog NM_001005862.1 2, neuro/ glioblastomaderived oncogene homolog (avian) CEA Carcinoembryonic NM_001712.4***NP_001703.2*** antigen EGFR epidermal growth NM_005228.3** NP_005219.2**factor receptor EphB4 EPH receptor B4 NM_004444.4 NP_004435.3 *MUC-1 hasa number of variants, any of which can be used in the methods describedherein. The sequence provided is variant 1. **EGFR has a number ofvariants, any of which can be used in the methods described herein. Thesequence provided is isoform a. ***CEA is a family of genes; any can beused here. The present sequences are for isoform 1 of CEACAM1.

Cancer Cell Surface Marker-Binding Agents

In some embodiments, the methods include the use of agents that bind tothe one or more cancer cell surface markers, e.g., bind specifically tothe markers. Any moiety that binds with sufficient specificity can beused. In some embodiments, antibodies (or antigen fragments thereof)that bind to the one or more cadherins are used. The term “antibody” asused herein refers to an immunoglobulin molecule or an antigen-bindingportion thereof. Examples of antigen-binding portions of immunoglobulinmolecules include F(ab) and F(ab′)2 fragments, which retain the abilityto bind antigen. The antibody can be polyclonal, monoclonal,recombinant, chimeric, de-immunized or humanized, fully human,non-human, (e.g., murine), or single chain antibody. In some embodimentsthe antibody has effector function and can fix complement. In someembodiments, the antibody has reduced or no ability to bind an Fcreceptor. For example, the antibody can be an isotype or subtype,fragment or other mutant, which does not support binding to an Fcreceptor, e.g., it has a mutagenized or deleted Fc receptor bindingregion. Methods for making antibodies and fragments thereof are known inthe art, see, e.g., Harlow et. al., editors, Antibodies: A LaboratoryManual (1988); Goding, Monoclonal Antibodies: Principles and Practice,(N.Y. Academic Press 1983); Howard and Kaser, Making and UsingAntibodies: A Practical Handbook (CRC Press; 1st edition, Dec. 13,2006); Kontermann and Dühel, Antibody Engineering Volume 1 (SpringerProtocols) (Springer; 2nd ed., May 21, 2010); Lo, Antibody Engineering:Methods and Protocols (Methods in Molecular Biology) (Humana Press; Nov.10, 2010); and Dühel, Handbook of Therapeutic Antibodies: Technologies,Emerging Developments and Approved Therapeutics, (Wiley-VCH; 1 editionSep. 7, 2010).

A number of antibodies that bind to the cancer cell surface markersdescribed herein are commercially available, e.g., from one or more ofthe following suppliers: Biorbyt, R&D Systems, Abcam, Bioss Inc.,Fitzgerald, Raybiotech, Genway, Abbiotec, Life Technologies, EMDMillipore, R&D Systems, Lifespan Biosciences, Thermo Fisher Scientific,Inc., AbD Serotec, ACris Antibodies, GenScript, Cell SignalingTechnology, OriGene, Novus Biologicals, Epitomics, Abgent, Aviva SystemsBiology, Abnova, ProSci, Biovisoin, GeneTex, and/or Uscn.

In some embodiments, protein binding partners are used. Some exemplarycadherin protein binding partners that can be used in the presentmethods and devices include ankyrin-1, beta-catenin, and FGFR; eitherthe full length or a fragment that binds to the cadherin can be used.

In some embodiments, nucleic acid based binding partners called aptamersare used. Nucleic acids can be selected to bind selectively to cadherinprotein targets; see, e.g., Ellington, A. D. and J. W. Szostak, Nature,346(6287):818-22 (1990); Gupta, S. et al., Appl Immunohistochem MolMorphol, 19(3):273-8 (2011).

In some embodiments, the binding agent can be coupled to a detectable orimaging agent. Detectable agents are well known in the art and includeparamagnetic agents, bioluminescent or fluorescent labels (e.g., GFP,FITC, rhodamine, or Texas Red), radioactive isotopes, andcolorimetric/enzymatic agents (e.g., HRP, B-galactosidase).

In some embodiments, the binding agent is coupled to a surface, e.g., asurface in a microfluidic device as described herein, a surface of anarray (e.g., a microarray), or a bead. In a preferred embodiment, theantibody is coupled to a magnetic bead, e.g., a paramagneticnanoparticle, e.g., cross-linked iron oxide (CLIO) nanoparticles; see,e.g., US 20110046004; Josephson et al., Bioconjug. Chem., 10(2):186-91(1999). Other devices can also be used, e.g., the CellSearch (FDA Finalrule. Fed Regist 69: 26036-26038 (2004); MagSweeper, Talasaz et al.,Proc Natl Acad Sci USA (2009) 106: 3970-3975; or NanostructuredSubstrates, Wang et al., Angew Chem Int Ed Engl 50: 3084-3088 (2011).These devices can be modified for use with the present methods.

Microfluidic Devices

In some embodiments, microfluidic (e.g., “lab-on-a-chip”) devices areused in the present methods. Such devices have been successfully usedfor microfluidic flow cytometry, continuous size-based separation, andchromatographic separation. In general, methods in which expression ofthe biomarkers is detected in circulating tumor cells (CTCs) can be usedfor the early detection of cancer, e.g., early detection of tumors ofepithelial origin, e.g., pancreatic, lung, breast, prostate, renal,ovarian or colon cancer.

The devices can be used for separating CTCs from a mixture of cells, orpreparing an enriched population of CTCs. In particular, such devicescan be used for the isolation of CTCs from complex mixtures such aswhole blood.

A variety of approaches can be used to separate CTCs from aheterogeneous sample. For example, a device can include an array ofmultiple posts arranged in a hexagonal packing pattern in a microfluidicchannel upstream of a block barrier. The posts and the block barrier canbe functionalized with different binding moieties. For example, theposts can be functionalized with one or more antibodies that bind to a(one or more) cadherin cancer cell surface marker as described herein tocapture circulating tumor cells (CTCs); see, e.g., Nagrath et al.,Nature 450:1235-1239 (2007), optionally with downstream block barriersfunctionalized with to capture biomarker nucleic acids or proteins.

Processes for enriching specific particles from a sample are generallybased on sequential processing steps, each of which reduces the numberof undesired cells/particles in the mixture, but one processing step maysuffice in some embodiments. Devices for carrying out various processingsteps can be separate or integrated into one microfluidic system. Thedevices include devices for cell/particle binding, devices for celllysis, devices for arraying cells, and devices for particle separation,e.g., based on size, shape, and/or deformability or other criteria. Incertain embodiments, processing steps are used to reduce the number ofcells prior to introducing them into the device or system. In someembodiments, the devices retain at least 75%, e.g., 80%, 90%, 95%, 98%,or 99% of the desired cells compared to the initial sample mixture,while enriching the population of desired cells by a factor of at least100, e.g., by 1000, 10,000, 100,000, or even 1,000,000 relative to oneor more non-desired cell types.

Some devices for the separation of particles rely on size-basedseparation with or without simultaneous cell binding. Some size-basedseparation devices include one or more arrays of obstacles that causelateral displacement of CTCs and other components of fluids, therebyoffering mechanisms of enriching or otherwise processing suchcomponents. The array(s) of obstacles for separating particles accordingto size typically define a network of gaps, wherein a fluid passingthrough a gap is divided unequally into subsequent gaps. Both sieve andarray sized-based separation devices can incorporate selectivelypermeable obstacles as described above with respect to cell-bindingdevices.

Devices including an array of obstacles that form a network of gaps caninclude, for example, a staggered two-dimensional array of obstacles,e.g., such that each successive row is offset by less than half of theperiod of the previous row. The obstacles can also be arranged indifferent patterns. Examples of possible obstacle shapes and patternsare discussed in more detail in WO 2004/029221.

In some embodiments, the device can provide separation and/or enrichmentof CTCs using array-based size separation methods, e.g., as described inU.S. Pat. Pub. No. 2007/0026413. In general, the devices include one ormore arrays of selectively permeable obstacles that cause lateraldisplacement of large particles such as CTCs and other componentssuspended in fluid samples, thereby offering mechanisms of enriching orotherwise processing such components, while also offering thepossibility of selectively binding other, smaller particles that canpenetrate into the voids in the dense matrices of nanotubes that make upthe obstacles. Devices that employ such selectively permeable obstaclesfor size, shape, or deformability based enrichment of particles,including filters, sieves, and enrichment or separation devices, aredescribed in International Publication Nos. 2004/029221 and 2004/113877;Nagrath et al., Nature 2007, 450:1235-1239; Huang et al. Science304:987-990 (2004), U.S. Publication No. 2004/0144651, U.S. Pat. Nos.5,837,115 and 6,692,952, and U.S. Application Nos. 60/703,833,60/704,067, and Ser. No. 11/227,904; devices useful for affinitycapture, e.g., those described in International Publication No.2004/029221 and U.S. application Ser. No. 11/071,679; devices useful forpreferential lysis of cells in a sample, e.g., those described inInternational Publication No. 2004/029221, U.S. Pat. No. 5,641,628, andU.S. Application No. 60/668,415; devices useful for arraying cells,e.g., those described in International Publication No. 2004/029221, U.S.Pat. No. 6,692,952, and U.S. application Ser. Nos. 10/778,831 and11/146,581; and devices useful for fluid delivery, e.g., those describedin U.S. application Ser. Nos. 11/071,270 and 11/227,469. Two or moredevices can be combined in series, e.g., as described in InternationalPublication No. WO 2004/029221. All of the foregoing are incorporated byreference herein.

In some embodiments, a device can contain obstacles that include bindingmoieties, e.g., monoclonal antibodies or antigen-binding fragmentsthereof, that selectively bind to cadherin cancer cell surface markers,e.g., on particular cell types, e.g., tumor cells. All of the obstaclesof the device can include these binding moieties; alternatively, only asubset of the obstacles includes them. Devices can also includeadditional modules, e.g., a cell counting module or a detection module,which are in fluid communication with the microfluidic channel device.For example, the detection module can be configured to visualize anoutput sample of the device.

In one example, a detection module can be in fluid communication with aseparation or enrichment device. The detection module can operate usingany method of detection disclosed herein, or other methods known in theart. For example, the detection module includes a microscope, a cellcounter, a magnet, a biocavity laser (see, e.g., Gourley et al., J.Phys. D: Appl. Phys., 36: R228-R239 (2003)), a mass spectrometer, a PCRdevice, an RT-PCR device, a microarray, or a hyperspectral imagingsystem (see, e.g., Vo-Dinh et al., IEEE Eng. Med. Biol. Mag., 23:40-49(2004)). In some embodiments, a computer terminal can be connected tothe detection module. For instance, the detection module can detect alabel that selectively binds to cells, proteins, or nucleic acids ofinterest, e.g., DNA, mRNA, or protein for a cadherin cancer cell surfacemarker as described herein.

In some embodiments, the microfluidic system includes (i) a device forseparation or enrichment of CTCs; (ii) optionally a device for lysis ofthe enriched CTCs; and (iii) a device for detection of DNA, mRNA, orproteins, e.g., DNA, mRNA, or protein for a cadherin cancer cell surfacemarker as described herein.

Other microfluidic platforms have been described; see, e.g., Sun et al.,Cancer Res 2010, 70:6128-6138.

In some embodiments, a device for affinity-based particle capture inmicrofluidic devices having grooves is used. A micro-channel formed in amicrofluidic device can be treated to capture particles suspended in afluid flowing through the channel. A particle capture efficiency of themicrofluidic device can be defined as a ratio of a number of particlescaptured in the channel and a total number of particles flowed throughthe channel. Grooves are formed extending into the walls of themicro-channel to create flow patterns in the fluid that promote aninteraction between the particles suspended in the fluid and innersurfaces of the walls of the channel. The increased interaction can leadto an increase in a number of particles captured in the channel, andconsequently, in the particle capture efficiency of the microfluidicdevice. The efficiency can further be increased by tailoring structuralfeatures of the microfluidic device including, for example, devicesubstrate material, channel and groove dimensions, and the like, as wellas fluid flow parameters such as flow rates based on types of particlesand the types of fluids in which the particles are suspended. In someembodiments, the grooves are arranged in a herringbone pattern formed byarranging grooves in a column in the micro-channel. Particles arecaptured in the micro-channel of the microfluidic device by forminggrooves in a wall of the micro-channel, coating an adherent on the innersurfaces of the walls of the micro-channel, and flowing particlessuspended in the fluid through the micro-channel. In suchimplementations, the adherent can be an antibody, for example, anantibody to a cadherin as described herein. See, e.g., the herringbonedevices described in Stott et al. PNAS 107(43):18392-7 (2010); and WO2010/036912.

In some embodiments, a device can separate and focus streams ofparticles to equilibrium positions within a channel flow field based, atleast in part, on inertial lift forces. In rectangular channels, thiscan lead, for example, to four streams of focused particles spaced adistance apart from a center of each of the four rectangular faces. Forcertain rectangular geometries, this four-fold symmetry can be reducedto a two-fold symmetry, with streams of particles spaced apart from eachof two opposed faces of the channel. Methods and structures thatdecrease the symmetry of the system using a variety of forces,including, for example, electromagnetic, magnetic, centrifugal,hydrodynamic, thermal, sonic, optical, and/or dielectrophoretic forcesor combinations thereof can be used. Although any force may be used tobias a particular potential minimum within the channel flow field,utilizing centrifugal forces with a curved channel structure has certainadvantages. In this case, the force will increase with the square of theflow rate based only on a minor geometric change with no additionalmechanical or electrical parts required. For example, the symmetry maybe reduced by using inertial forces inherent in the flow through anS-shaped rectangular channel to result in a two-fold symmetry (down fromfour-fold) with a majority of the particles aligned with the flow in aperiodic manner not corresponding to the period of the underlyingchannel. The geometry of the channel may also be used to change symmetryeither by changing the radius of curvature or the width of the channelin a periodic manner (the channels thus curving asymmetrically) tocreate a single focused particle stream. See, e.g., WO 2008/130977.

Devices can include one or more arrays of obstacles that allow lateraldisplacement of circulating tumor cells and other components of fluids,thereby offering mechanisms of enriching or otherwise processing suchcomponents. Devices that employ obstacles for this purpose aredescribed, e.g., in Huang et al. Science 304, 987-990 (2004) and U.S.Publication No. 20040144651. The devices for separating particlesaccording to size typically employ an array of a network of gaps,wherein a fluid passing through a gap is divided unequally intosubsequent gaps. The array can include a network of gaps arranged suchthat fluid passing through a gap is divided unequally, even though thegaps may be identical in dimensions. The method uses a flow that carriescells to be separated through the array of gaps. The flow is aligned ata small angle (flow angle) with respect to a line-of-sight of the array.Cells having a hydrodynamic size larger than a critical size migratealong the line-of-sight, i.e., laterally, through the array, whereasthose having a hydrodynamic size smaller than the critical size followthe average flow direction. Flow in the device occurs under laminar flowconditions. Devices are optionally configured as continuous-flowdevices. See, e.g., U.S. Pat. No. 7,988,840.

In some embodiments, the present invention includes devices (e.g.,microfluidic devices) that contain a plurality of magnetic particles. Bycoating these magnetic particles with appropriate capture moieties,e.g., monoclonal antibodies or antigen-binding fragments thereof thatbind to cancer cell surface markers as described herein (e.g., one ormore cadherins, optionally with additional markers), it is possible toselectively bind circulating tumor cells through affinity mechanisms. Insome embodiments, the magnetic particles are fixed obstacles set in achannel in a device. In its simplest embodiment, the device includes achannel having magnetic regions to which magnetic particles canmagnetically attach to create a textured surface, with which cellspassing through the channel can come into contact. The magneticparticles can serve to texture the channel, and through the appropriatechoice of magnetic particle size and shape relative to the dimensions ofthe channel, it is possible to provide a texture that enhancesinteractions between the cells of interest and the magnetic particles.The magnetic particles can be magnetically attached to hard magneticregions of the channel or to soft magnetic regions that are actuated toproduce a magnetic field. In addition, these magnetic particles can bereleased from defined locations within the channel, e.g., by increasingthe overall flow rate of the fluid flowing through the device,decreasing the magnetic field, or through some combination of the two.In some embodiments, a spatially nonuniform permanent magnet orelectromagnet may be used to create organized and in some cases periodicarrays of magnetic particles within an otherwise untextured microfluidicchannel (Deng et al. Applied Physics Letters, 78, 1775 (2001)). Anelectromagnetic may be employed to create a non-uniform magnetic fieldin a device. The non-uniform filed creates regions of higher and lowermagnetic field strength, which, in turn, will attract magnetic particlesin a periodic arrangement within the device. Other external magneticfields may be employed to create magnetic regions to which magneticparticles attach. A hard magnetic material may also be used in thefabrication of the device, thereby obviating the need for electromagnetsor external magnetic fields. In one embodiment, the device contains aplurality of channels having magnetic regions, e.g., to increasevolumetric throughput. Further, these channels may be stackedvertically. The devices described herein can all include a spiralchannel that is designed to apply differential inertial focusing to theblood sample. See, e.g., US20100055758.

In some embodiments, a device can contain one or more structures thatdeterministically deflect particles, in a fluid, having a hydrodynamicsize above a critical size in a direction not parallel to the averagedirection of flow of the fluid in the structure. An exemplary structureincludes an array of obstacles that form a network of gaps, wherein afluid passing through the gaps is divided unequally into a major fluxand a minor flux so that the average direction of the major flux is notparallel to the average direction of fluidic flow in the channel, andthe major flux from the first outer region is directed either toward thesecond outer region or away from the second outer region, wherein theparticles are directed into the major flux. The array of obstaclespreferably includes first and second rows displaced laterally relativeto one another so that fluid passing through a gap in the first row isdivided unequally into two gaps in the second row. Such structures maybe arranged in series in a single channel, in parallel in the samechannel, e.g., a duplex configuration, in parallel in multiple channelsin a device, or combinations thereof. Each channel will have at leastone inlet and at least one outlet. A single inlet and outlet may beemployed for two or more structures in parallel, in the same ordifferent channels. Alternatively, each structure may have its own inletand outlet or a single structure may contain multiple inlets andoutlets, e.g., to introduce or collect two different fluidssimultaneously. See, e.g., U.S. Pat. No. 8,021,614.

Also included herein are any and all of the above-described devices,having disposed therein an agent that binds a cancer cell surface markerselected from the group consisting of cadherin 1 (CDH1), CDH2, CDH3,CDH4, CDH5, CDH9, CDH11, CDH17, CDH19, protocadherin 9 (PCDH9) and/orPCDH beta 13 (PCDHb13), plus one or more additional cancer cell surfacemarkers, e.g., EpCAM. In some embodiments, the devices comprise agentsthat bind to cadherin 3 and agents that bind to cadherin 11. In someembodiments, the devices comprise agents that bind to cadherin 3, agentsthat bind to cadherin 11, and agents that bind to EpCAM.

Surface-based Separation Methods

In some embodiments, the methods and devices include the use ofsurfaces, e.g., arrays or beads, e.g., magnetic beads, that are coatedwith a binding agent as described herein to allow the surfaces to bindspecifically to cells expressing a cadherin cancer cell surface markeras described herein. In some embodiments, the methods can include theuse of magnetic beads coated with one or more binding agents, e.g.,antibodies or antigen-binding fragments thereof, that bind to a cadherincancer cell surface marker as described herein. In some embodiments,each bead is coated with antibodies that bind to only one cadherincancer cell surface marker as described herein; in some embodiments,each bead is coated with antibodies to a plurality of cadherin markers,such that each bead can bind to more than one cadherin cancer cellsurface marker as described herein. In this way, the cancer cells are“labeled” with the beads. A number of magnetic beads are known in theart and can be used for the methods described herein. For example, latexbeads that include an iron oxide component, e.g., as a core or admixedinto the latex, can be used. Methods for making magnetic beads are knownin the art; see, e.g., US2006/0154309, Marik et al., Journal ofMagnetism and Magnetic Materials, 264(2-3):153-157 (2003).

Cancer cells labeled with the beads or bound to a surface can becaptured from the sample using methods known in the art. For example, inthe case of magnetic beads, magnetic cell separation methods as known inthe art can be used, e.g., as described in U.S. Pat. No. 5,514,340,US20110059500, US2006/0154309, US 2010/0159556, and PCT US/93/04145.

Examples

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Identification of Candidate Cancer Cell Surface Markers

To identify potential candidate cancer cell surface markers, RNAexpression in human CTC samples was analyzed. A herringbone CTC capturedevice (^(Hb)CTC-chip; see, e.g., Stott et al. PNAS 107(43):18392-7(2010); WO 2010/036912) was used to capture CTCs from 12 metastaticpancreatic cancer patients. RNA was extracted from the ^(Hb)CTC-chip aswell as a paired control non-capture IgG herringbone device. The controldevice allowed for the ability to extract genes upregulated in CTCs.

Four cadherin family members were identified in this preliminary screen,as shown in Table 1.

TABLE 1 CDH4 cadherin 4, type 1, R-cadherin (retinal) CDH9 cadherin 9,type 2 (T1-cadherin) PCDH9 protocadherin 9 PCDHB13 protocadherin beta 13

Example 2 Cadherin Expression in CTCs

A targeted search for cadherins expressed in CTCs was undertaken. Theresults of the search identified CDH4 and CDH9 as potential cadherinsexpressed in CTCs. CDH4 was analyzed further in cell line models, whichrevealed expression in the highly metastatic breast cancer cell lineMDA-MB231 by Western blot (FIG. 1).

Immunofluorescent staining confirmed expression of CDH4 in this cellline, but almost no expression of EpCAM despite its highly metastaticphenotype (FIG. 2). Therefore, this particular cancer cell line wouldnot be captured on current CTC devices based on EpCAM, but would becaptured with an anti-CDH4 device.

A more comprehensive bioinformatics analysis of cadherins was then takenin primary and secondary tumor expression databases. Candidates wereevaluated for the presence of significant levels of expression intumors, but near absence in healthy donor whole blood analytes in thedatabase (see FIGS. 3-49). Cadherin candidates were then refined furtherby using the Oncomine™ database to identify potential CTC captureantigens (FIG. 2).

In summary, cadherins represent novel CTC capture antigens obtained fromRNA sequencing in pancreatic CTCs. CDH4 is expressed on cancer celllines that do not express EpCAM and therefore a viable capture marker.

More extensive analysis of primary tumors evaluated by gene expressionmicroarrays has also identified CDH 1, 2, 3, 5, 11, and 19 as promisinghighly expressed cadherins in a wide variety of tumors but nearly absentin normal blood.

Example 3 Cadherins Capture CTCs In Vivo

The ability to capture CTCs from an experimental animal implanted withhuman cancer cells was determined. Cadherin expression in normal breastepithelial cells (NBE), CD44+ cells isolated from pleural effusions(P_CD44+) of human breast cancer patients, and CD44+(X_CD44+) and CD44−(X_CD44−) cells isolated from tumor xenografts grown in mice.

The results are shown in FIGS. 50A-50D, and demonstrate that thedifferential expression of cadherins in tumor cell subfractions relativeto that observed in normal breast epithelial cells.

TGF-beta treatment of epithelial cells induces anepithelial-to-mesenchymal transition (EMT). As shown in FIGS. 51A-F,some cadherins increase, others decrease and some do not change duringEMT, providing further evidence that a cocktail of cadherins wouldcapture both epithelial and mesenchymal tumor cells.

Example 4 Analysis of Cadherin Expression

First, Western blotting was used to analyze expression of EpCAM andcadherins 1, 2, 3, 5, 11, and 17, in various cancer cell lines,including the following: Breast: MCF10A, SKBR3, MDA-MB-231*,(MCF10A-LBX1); Prostate: PC3-9; Lung: H1650, CaLu1*, HCC827; and Colon:SW620, HCT116.

Briefly, total protein isolated from above cells lines was separated bySDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes andanalyzed using antibodies against EPCAM and cadherins 1, 2, 3, 5, 11,and 17.

The summary of western blot results shown in Table 2, demonstrateswidespread expression of Cadherins, especially CDH3 and CDH11, and thatexpression of CDH3 and CDH11 complement expression of EpCAM. It is wellestablished that antibody reactivity towards proteins varies dependingon the application used. For example, some antibodies work better onwestern blot than with FACS sorting. Hence several antibodies weretested to ensure specific detection.

FACS analysis was used to further determine the ability of panels ofanti-cadherin antibodies, with and without EpCAM, to capture tumorcells. This example provides evidence that a cocktail of antibodiesagainst cell surface proteins, e.g., the epithelial-specific EpCAM andthe epithelial and mesenchyme specific-Cadherins, will capture theentire spectrum of CTCs of different lineages.

Cells were cultured to 70-80% confluence and released from the culturedish using cell dissociation solution. Cells (250,000) were resuspendedin 300 μl of PBS and 100 μl of antibody against each protein (cadherin 3(AbCAM), 11 (R&D), EpCAM (Veridex)), either alone or in combination, for30 minutes at 4 degrees. Isotype matched IgG was used as control. Afterincubation with appropriate secondary antibodies (30 minutes, 4degrees), the cells were washed and analyzed by FACS. Over all about10,000 cells were sorted to obtain the data provided.

The data, shown in Table 3, demonstrates that an antibody cocktailagainst EpCAM and cadherins 3 and 11 captures cancer cell lines of bothepithelial and mesenchymal phenotypes at nearly ˜90%. [10 out of 10 celllines (n=2).

Thus, combining antibodies against CDH3 and CDH11 with an EpCAM antibodywas able to detect 90% of the tumor cells irrespective of theirepithelial or mesenchymal state (it should be noted that MDA-MB-231,CaLu1 and PC3-9 cells express lower levels of EpCAM and that MDA-MB-231and CaLu1 are highly mesenchymal in culture). Thus, sorting with panelsincluding antibodies to CDH11 and EpCAM, or to CDH3, CDH11, and EpCAM,captured nearly or greater than 90% of cells in a wide variety of celltypes.

FACS was further used to determine whether antibodies to CDH3 and CDH11would also bind to whole blood or white blood cells. The results, shownin FIGS. 52A-B, shown that while Abcam 89900 anti-CDH3 antibodies boundrobustly to lung cancer H1650 cells (FIG. 52A, left panel), and RnD 1790anti-CDH11 antibodies bound to PC3-9 prostate cancer cells (FIG. 52B,left panel), neither antibody bound significantly to white blood cells(52A-B, middle panels) or whole blood (52A-B, right panels).

When the expression results were compared between FACS and Westernblotting, excellent concordance was seen with several of the antibodiestested (Table 4).

Finally, the ability of a combination of CDH3 and CDH11 antibodies tocapture cancer cells in a mixture of cells was tested. Calu1 breastcancer cells and H1650 lung cancer cells were mixed and contacted withanti-CDH3 Abcam 89900 antibodies and RnD anti-CDH11 1790 antibodies. Theresults, shown in FIG. 53 and Table 5, demonstrate that this combinationwas extremely effective in capturing nearly all of the cancer cells.

TABLE 2 SKBR3 H1650 PC3-9 MB-231 LBX1 MCF10A HCC827 Calu-1 SW620 HCT116(E) CDH1 Abcam 1416 + + + + + (E) CDH1 Cell Signaling 3195 + + + + + (E)CDH1 BD Transduction 610181 + + + + + CDH2 eBioscience 14325982 + (N)CDH2 Invitrogen 333900 + + + (N) CDH2 Abcam 124397 + + + (P) CDH3 BDTransduction 610227 + + + + (P) CDH3 Abcam12222 (P) CDH3 Abcam89900 + + + + + (P) CDH3 RnD MAB861 + + + + + (VE) CDH5 Abcam 7047multiple bands (VE) CDH5 Cell Signaling 2500 + CDH11 Millipore MAB2014CDH11 Invitrogen 717600 + + + + + + + + + + CDH11 Invitrogen321700 + + + CDH11 RnD MAB1790 CDH17 RnD BAF1032 CDH17 Abcam 124266CDH17 Abcam 89305 CDH17 AbD Serotec EpCAM RnD BAF960 + + + + + + EpCAMVeridex + + + + + +

TABLE 3 Lung Colon Prostate Breast Br Ctrl CaLu1 H1650 HCC827 HCT116SW620 PC39 MCF10A SKBR3 MB231 LBX1 EpCAM Veridex *19.5 98.5 95.4 94.9 93*70.8 93.9 98.9 *75.5 *9.5 CDH3 Abcam 89900 *0.8 95.9 87.4 99.7 *1.7*1.6 93.9 *0.5 *4.4 56.2 CHD11 RnD 1790 92.9 *10.9 *13.9 *10.4 *2.4 94*6.7 *17.7 93.8 41 CDH3 & CDH11 91 63.6 67.9 99.1 2.3 92.5 93.8 9.6 90.650.5 EpCAM & CDH3 19.2 97.5 87.5 85.7 89.9 86.2 97.2 83.6 72.1 47 EpCAM& CDH11 93.5 97.5 94.9 99.7 98.8 96.9 96.4 98.8 95.8 13.6 EpCAM & CDH3&CDH11 92.9 98.5 *88.9 99.1 *78.5 97.6 97.6 98.9 95.3 45.4 mslgG2b 0.30.3 0.3 2 0.3 0.3 0.4 0.3 0.3 0.3 mslgG1 0.3 0.9 0.3 2 0.3 0.3 0.4 0.30.3 0.3 mslgG1 0.3 0.8 0.3 2 0.3 0.3 0.4 0.3 0.3 0.3 mslgG1 &mslgG2b 0.30.3 0.3 2 0.3 0.3 0.4 0.3 0.3 0.3 mslgG1 &mslgG2b 0.3 0.5 0.3 2 0.3 0.30.4 0.3 0.3 0.3

TABLE 4 Western Blotting Calu-1 H1650 HCC827 HCT116 SW620 PC3-9 MCF10ASKBR3 MB-231 LBX1 (P) CDH3 Abcam + + + + + (P) CDH3 R&D + + + + + CDH11Invitrogen + + + EpCAM RnD + + + + + + EpCAM Veridex + + + + + + FACSCalu1 H1650 HCC827 HCT116 SW620 PC39 MCF10A SKBR3 MB-231 LBX1 CDH3 Abcam0.8 95.9 87.4 99.7 1.7 1.6 93.9 0.5 4.4 56.2 CHD11 R&D 92.9 10.9 13.910.4 2.4 94 6.7 17.7 93.8 41 EpCAM Veridex 19.5 98.5 95.4 94.9 93 70.893.9 98.9 75.5 9.5 CDH3 & CDH11 91 63.6 67.9 99.1 2.3 92.5 93.8 9.6 90.650.5 EpCAM & CDH3 19.2 97.5 87.5 85.7 89.9 86.2 97.2 83.6 72.1 47 EpCAM& CDH11 93.5 97.5 94.9 99.7 98.8 96.9 96.4 98.8 95.8 13.6 EpCAM & CDH3&CDH11 92.9 98.5 88.9 99.1 78.5 97.6 97.6 98.9 95.3 45.4

TABLE 5 Lung Calu1 H1650 Calu1 + H1650 CDH3 (Abcam 89900) 0.9 99.5 60.5CHD11 (RnD 1790) 94.2 8.3 41.9 CDH3 & CDH11 N/A N/A 93.2

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of detecting the presence of acirculating tumor cell (CTC) in a sample from a subject, the methodcomprising: providing a sample comprising blood from a subject; andcontacting the sample with one or more agents that bind to a cancer cellsurface marker selected from the group consisting of cadherin 1 (CDH1),CDH2, CDH3, CDH4, CDH5, CDH9, CDH11, CDH17, CDH19, protocadherin 9(PCDH9) and/or PCDH beta 13 (PCDHb13); and detecting the binding of theagent to a cancer cell surface marker present on a cell in the sample;thereby detecting the presence of a cancer cell in the sample.
 2. Amethod of capturing a circulating tumor cell (CTC) from a sample, themethod comprising: providing a sample comprising blood from a subject;and contacting the sample with one or more agents that bind to a cancercell surface marker selected from the group consisting of cadherin 1(CDH1), CDH2, CDH3, CDH4, CDH5, CDH9, CDH11, CDH17, CDH19, protocadherin9 (PCDH9) and/or PCDH beta 13 (PCDHb13), under conditions sufficient toallow binding of the agent to cells expressing the surface marker; andisolating a cell expressing a cancer cell surface marker from thesample; thereby capturing a CTC from the sample.
 3. A method ofdiagnosing cancer in a subject, the method comprising: providing asample comprising blood from the subject; and contacting the sample withone or more agents that bind to a cancer cell surface marker selectedfrom the group consisting of cadherin 1 (CDH1), CDH2, CDH3, CDH4, CDH5,CDH9, CDH11, CDH17, CDH19, protocadherin 9 (PCDH9) and/or PCDH beta 13(PCDHb13), under conditions sufficient to allow binding of the agent tocells expressing the surface marker; and detecting the binding of theagent to a cancer cell surface marker present on a cell in the sample;wherein the presence of binding to a cell in the sample indicates thatthe subject has cancer.
 4. A method of monitoring development orprogression of cancer in a subject, the method comprising: providing afirst sample comprising blood from the subject; and contacting the firstsample with one or more agents that bind to a cancer cell surface markerselected from the group consisting of cadherin 1 (CDH1), CDH2, CDH3,CDH4, CDH5, CDH9, CDH11, CDH17, CDH19, protocadherin 9 (PCDH9) and/orPCDH beta 13 (PCDHb13), under conditions sufficient to allow binding ofthe agent to cells expressing the surface marker; and detecting thepresence or absence of binding of the agent to a cancer cell surfacemarker present on a cell in the first sample; providing a subsequentsample comprising blood from the subject; and contacting the subsequentsample with one or more agents that bind to a cancer cell surface markerselected from the group consisting of cadherin 1 (CDH1), CDH2, CDH3,CDH4, CDH5, CDH9, CDH11, CDH17, CDH19, protocadherin 9 (PCDH9) and/orPCDH beta 13 (PCDHb13), under conditions sufficient to allow binding ofthe agent to cells expressing the surface marker; and detecting thepresence or absence of binding of the agent to a cancer cell surfacemarker present on a cell in the subsequent sample.
 5. The method ofclaim 4, wherein the absence of binding to a cell in both the first andsubsequence sample indicates that the subject has not developed cancer.6. The method of claim 4, wherein the absence of binding to a cell inthe first sample, and the presence of binding to a cell in a subsequencesample indicates that the subject has developed cancer.
 7. The method ofclaim 4, further comprising quantifying a level of surfacemarker-expressing cells in the first and subsequent samples, wherein anincrease in the number of cells that express the one or more surfacemarkers indicates that cancer is progressing in the subject; a decreasein the number of cells that express the one or more surface markersindicates that cancer is regressing in the subject; and no significantchange in the number of cells that express the one or more surfacemarkers indicates that cancer is stable in the subject.
 8. The method ofany of claims 1-4, further comprising contacting the sample with one ormore agents that bind to an additional cancer cell surface markerselected from the group consisting of EpCAM, MUC-1, HER2, EGFR, EphB4,and CEA.
 9. The method of claim 8, wherein the additional cancer cellsurface marker is EpCAM.
 10. The method of any of claims 1-9, whereinthe one or more agents that bind to a cancer cell surface marker are ina microfluidic device.
 11. The method of any of claims 1-10, wherein theone or more agents that bind to a cancer cell surface marker are coatedon a magnetic bead.
 12. The method of any of claims 1-11, wherein theone or more agents that bind to a cancer cell surface marker areantibodies or antigen-binding fragments thereof.
 13. The method of anyof claims 1-12, wherein the one or more agents that bind to a cancercell surface marker comprise agents that bind to CDH1, 2, 3, 5, and/or11.
 14. The method of any of claims 1-12, wherein the one or more agentsthat bind to a cancer cell surface marker comprise agents that bind toCDH3 and/or CD11.
 15. The method of any of claims 1-14, wherein themethod is performed using a microfluidic device.
 16. The method of claim15, wherein the microfluidic device separates tumor cells based oninertial lift forces or fluid flow patterns.
 17. A device for separationof cells in a sample, the device comprising an inlet, and outlet, andone or more areas coated with an agent that binds a cancer cell surfacemarker selected from the group consisting of cadherin 1 (CDH1), CDH2,CDH3, CDH4, CDH5, CDH9, CDH11, CDH17, CDH19, protocadherin 9 (PCDH9)and/or PCDH beta 13 (PCDHb13), configured to separate cells that expressthe cancer cell surface marker from cells that do not express the cancercell surface marker.
 18. The device of claim 14, wherein the device is amicrofluidic device.
 19. The device of claim 14 or 15, wherein thedevice comprises a micro-channel disposed between the inlet and theoutlet, and a herringbone pattern formed by arranging grooves in themicro-channel, and the agent that binds the cancer cell surface markeris disposed on one or more of walls of the microchannel.
 20. The methodof claim 19, comprising one or more areas coated with an agent thatbinds to CDH4, one or more areas coated with an agent that binds toCDH9, or both.
 21. The method of claim 19, comprising one or more areascoated with an agent that binds to CDH3, one or more areas coated withan agent that binds to CD11, or both.
 22. The device of any of claims14-21, further comprising one or more areas coated with an agent thatbinds to an additional cancer cell surface marker selected from thegroup consisting of EpCAM, MUC-1, HER2, EGFR, EphB4, and CEA.
 23. Thedevice of claim 22, wherein the additional cancer cell surface marker isEpCAM.
 24. The device of claim 14, wherein the one or more agents thatbind to a cancer cell surface marker are coated on a magnetic bead. 25.The device of claims 14-24, wherein the one or more agents that bind toa cancer cell surface marker are antibodies or antigen-binding fragmentsthereof.